Retinal morphogenesis in zebrafish

描述（由申请方提供）：脉络膜裂（CF）闭合期间的畸形是视网膜形态发生的重要步骤，是先天性视力障碍的重要原因。Hh信号在CF形态发生过程中起着至关重要的作用;然而，在我们对控制这一过程的遗传和细胞机制的理解中存在根本性的差距。我们已经确定了一个新的作用，斑马鱼Zic 2a，一个保守的锌指转录因子，作为一个关键的调制器的Hh调节基因表达的前脑和视网膜，并证明了一个必不可少的作用，Zic 2a CF形态发生。我们研究的长期目标是阐明控制CF形态发生的基因调控网络。实现这一目标的一个重要步骤，Zic 2a效应子的鉴定和CF形成过程中其功能的详细分析，是本提案的目的。我们已经表明，Zic 2a在邻近CF的视柄中表达，并且非细胞自主地起作用以促进CF形成。基于这些数据，我们假设Zic 2a通过Hh信号传导来控制驱动OS和CF形态发生的差异细胞动力学，并且Zic 2a调节在OS/视网膜边界形成期间具有关键功能的基因的转录。该假设将通过追求两个特定目标来测试：（1）表征形成OS/视网膜边界处的细胞动力学并分析Hh信号和Zic功能在调节这些动力学中的作用;（2）鉴定视柄和CF中Zic 2a的转录靶，并测试它们在CF形态发生期间的作用。在第一个目标下，CF形态发生期间的细胞动力学将使用高分辨率实时成像进行检查，包括正常发育期间和Zic功能和Hh信号传导中断的胚胎中。在第二个目标下，将使用荧光激活分选从具有正常或破坏的Zic 2a表达的胚胎中分离视柄细胞，并且将使用高通量测序（RNA-seq）鉴定需要Zic 2a功能以在这些细胞中正确表达的基因。这些基因在CF形成过程中的功能将通过条件性过表达测定和用工程化锌指核酸酶的靶向诱变的组合来评估。这项工作意义重大，因为它将确定一个非常重要的遗传网络的新组成部分，该网络控制视网膜形态发生，并由Hedghog信号协调。这种方法是创新的，因为它集成了分子，遗传和细胞生物学方法在一个实验生物体，斑马鱼，检查CF形态发生过程中Zic 2a的新功能。这种方法将从根本上推进我们对一个重要的悬而未决的问题的理解：Hh信号如何在视网膜发育过程中协调图案化和形态发生，并将为分析Zics的其他重要但知之甚少的功能奠定坚实的基础，包括它们在神经管闭合，神经元再生和肿瘤发生中的作用。 公共卫生相关性：这项拟议的研究与人类健康有关，因为它针对一种保守但知之甚少的遗传机制，该机制控制视网膜形态发生的一个方面（脉络膜裂形成），特别容易在人类发育过程中受到破坏。这项工作将确定人类缺损的新候选基因，并将为小分子筛选奠定基础，以确定CF闭合的调节剂，最终目标是设计药理学干预措施，以减轻缺损相关的视力损害。

DESCRIPTION (provided by applicant): Malformations during choroid fissure (CF) closure, an important step in retinal morphogenesis, are a significant cause of congenital visual impairment. Hh signaling plays a crucial role during CF morphogenesis; however, there is a fundamental gap in our understanding of the genetic and cellular mechanisms that control this process. We have identified a novel role for zebrafish Zic2a, a conserved zinc-finger transcription factor, as a key modulator of Hh-regulated gene expression in the forebrain and retina, and demonstrated an essential role for Zic2a in CF morphogenesis. The long-term goal of our research is to elucidate the gene regulatory network that controls CF morphogenesis. An essential step toward this goal, identification of Zic2a effectors and detailed analysis of their functions during CF formation, is the objective of this proposal. We have shown that Zic2a is expressed in the optic stalk, which is adjacent to the CF, and functions non-cell-autonomously to promote CF formation. Based on these data, we hypothesize that Zic2a functions through Hh signaling to control differential cellular dynamics that drive OS and CF morphogenesis, and that Zic2a regulates transcription of genes with key functions during OS/retinal border formation. This hypothesis will be tested by pursuing two Specific Aims: (1) Characterize cellular dynamics at the forming OS/retinal boundary and analyze the role for Hh signals and Zic function in regulating these dynamics; (2) Identify transcriptional targets of Zic2a in the optic stalk and CF, and test their roles during CF morphogenesis. Under the first aim, cellular dynamics during CF morphogenesis will be examined using high-resolution real-time imaging, both during normal development and in embryos with disrupted Zic function and Hh signaling. Under the second aim, optic stalk cells will be isolated from embryos with normal or disrupted Zic2a expression using fluorescence-activated sorting, and genes that require Zic2a function for their correct expression in these cells will be identified using high-throughput sequencing (RNA-seq). Functions of these genes during CF formation will be assessed through a combination of conditional over expression assays and targeted mutagenesis with engineered zinc finger nucleases. The proposed effort is significant because it will identify new components of a very important genetic network that controls retinal morphogenesis and is coordinated by Hedghog signaling. This approach is innovative because it integrates molecular, genetic and cell biological approaches in one experimental organism, the zebrafish, to examine a novel function for Zic2a during CF morphogenesis. This approach will fundamentally advance our understanding of an important outstanding question: how Hh signaling coordinates patterning and morphogenesis during retinal development, and will build a strong foundation for analyses of other important but poorly understood functions of Zics, including their roles in neural tube closure, neuronal regeneration and tumorigenesis. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to human health because it targets a conserved but poorly understood genetic mechanism that controls an aspect of retinal morphogenesis (choroid fissure formation), particularly vulnerable to disruption during human development. This work will identify novel candidate genes for human coloboma and will set the stage for small-molecule screens to identify modulators of CF closure, with the ultimate goal of designing pharmacological interventions to alleviate coloboma-associated visual impairment.